RNA interference (RNAi) has become an important technique for specifically suppressing expression of a target gene in the fields of e.g., molecular biology, pharmacology and medical science. RNAi can be induced by introducing a short double-stranded RNA called siRNA (small interfering RNA) consisting of 20 to 23 nucleotides in a cell. The siRNA, although it is a small molecule, permeability of cell membrane to siRNA is insufficient and stability of siRNA in serum is insufficient. Because of this, the efficiency of inducing RNAi by siRNA still has room for improvement. In addition, siRNA has a problem in that siRNA activates natural immunity via a pattern recognition receptor such as Toll-like receptor.
The present inventors disclose in Patent Literature 1 a method for constructing a functional molecule in a cell by preparing a functional nucleic acid molecule such as siRNA in the form of being easily taken up by a cell and introducing the molecule in the cell (see, also Non-Patent Literature 1). This method is a construction method for a functional nucleic acid molecule consisting of one or two nucleic acid chains and comprises the following steps 1) and 2).
1) a step of introducing two or more fragments having functional groups, which mutually bind by a chemical reaction, at the corresponding ends thereof, in a cell, and
2) a step of reacting the above functional groups to bind the fragments in the cell to produce a functional nucleic acid molecule consisting of one or two nucleic acid chains.
In the above method, at least part of the nucleic acid chain constituting a functional nucleic acid molecule is divided into a plurality of fragments and the fragments are introduced in a cell to construct the functional nucleic acid molecule in the cell (hereinafter also referred to as an “intracellular built-up method”). In this method, an electrophilic group is attached to an end of one of the fragments and a nucleophilic group is attached to the end of another fragment corresponding to the aforementioned end, and the fragments are bound by the chemical reaction between these groups. More specifically, an iodoacetyl group, a bromoacetyl group or an iodo group is used as the electrophilic group and a phosphorothioate group is used as the nucleophilic group. These groups are chemically reacted to connect riboses of two fragments.